The aim of the project is to identify the intermediates involved in the elongation cycle of protein biosynthesis of E.coli and to determine the rate constants of their interconversion. This will result in an understanding of the role of GTP hydrolysis in the fast and accurate incorporation of amino acids into the growing peptide chain. This is important to the wider problem, that of understanding how the free energy of nucleoside triphosphate hydrolysis is used to promote biological processes involving protein - nucleic acid interactions. Mechanisms by which energy is utilized to ensure accurate information transfer, whether replication transcription or translation are fundamental to biology and their breakdown may well play a role in the development of cellular malfunction. Techniques used will be based on novel semi-micro rapid quenching methods and stopped-flow spectrophotometry following the introduction of suitable chromophoric or fluorescent probes into the system, together with the use of stable isotopes of oxygen to study the mechanism of GTP hydrolysis. Kinetics and equilibrium constants will be measured for the formation of the aminoacyl-tRNA.EF.Tu.GTP ternary complex, and binding of this complex to ribosomes, cleavage of GTP, formation of peptide, and release of products. These are the known steps in the elongation cycle. It is expected that other, as yet unknown, steps will be identified by the techniques used.